There are accumulated evidences that the extracellular matrix (ECM) not only acts as a support structure but also has considerable effects on neuronal development and regeneration, synaptic plasticity, neuronal excitability, and homeostatic regulations of network activity. Indeed, the ECM has a profound impact on network behavior, hence on physiological processes such as cognition. Gelatinases belong to the Matrixines family (MMPs), which is formed by zinc dependent endopeptidases capable to degrade ECM proteins as well as a large number of non-ECM proteins, such as growth factors, cytokines, chemokines, cell surface receptors, serine proteinase inhibitors and other MMPs. MMPs are either secreted or membrane bound proteases and play major physiological roles in reproduction, growth, development, angiogenesis, immune response, wound healing and brain physiology. There is enhanced expression of MMPs, in particular gelatinases (MMP-2 or gelatinase A and MMP-9 or gelatinase B), during numerous pathological conditions, including tumor progression, neurodegeneration, stroke, inflammation and viral infections.
Initial studies on gelatinase MMP-9 in the brain originally were focused on its possible role in a variety of pathological conditions with a degenerative component. More recently, MMP-9 has emerged as an important player in the brain physiology, especially as being a key molecule in the synaptic plasticity. These findings led to studies demonstrating a possible involvement of MMP-9 in neuropsychiatric conditions such as epilepsy, schizophrenia, Alzheimer disease, autism (in particular associated to fragile X syndrome), mental retardation, bipolar disorders, mood disorders such as bipolar disorders, depression and drug addiction.
Alterations in glutamate signaling, leading to aberrant synaptic plasticity, in schizophrenia have been described. MMP-9 has been shown to regulate glutamate receptors, and modulating physiological and morphological synaptic plasticity. By means of functional gene polymorphism, gene responsiveness to antipsychotics and blood plasma levels, MMP-9 has recently been related to schizophrenia. This disease involves impairments in perception and cognition, culminating in a triad of positive, negative, and cognitive symptoms that are believed to reflect modifications in neuronal circuitry, the perturbation of synaptic connectivity, and alterations in dendritic spines. Notably, chromosome region 20q11-13, where the MMP-9 gene is located, has been extensively studied in terms of psychiatric disorders and linked to schizophrenia. MMP-9 influences hippocampal and prefrontal cortical function and is an interesting candidate molecule that is potentially involved in schizophrenia, a condition in which prefrontal cortex impairment is one of the most common pathological findings. Furthermore, some of the target candidate proteins that are implicated in schizophrenia have functional connections with either MMP-9 or MMP-9-interacting proteins, such as brain derived neurotrophic factor (BDNF) and N-methyl-D-aspartate (NMDA) receptors, among others. Abnormal elevated MMP-9 levels have been found in plasma of schizophrenic patients as reported by several authors (Yamamori, H. et al., Neurosci Lett., 2013, (556):37-41).
There are accumulating evidences of the involvement of MMPs in the pathogenesis of epilepsy. This disease is a brain disorder characterized by an enduring predisposition to generate epileptic seizures and by the neurobiological, cognitive, psychological, and social consequences of this condition. It has been demonstrated that prolonged seizures are related to high-serum MMP-9 levels. More importantly, recent studies in brain tissue obtained during epilepsy surgery reported increased MMP-9 immunoreactivity in epileptogenic lesions of focal cortical dysplasia (Konopka, A. et al., Epilepsy Res., 2013, (1-2):45-58) and tuberous sclerosis (Li, S. et al., Brain Res, 2012, (1453):46-55) as well as in the epileptogenic cortical or hippocampal lesions of patients with temporal lobe epilepsy without underlying cytoarchitectonic abnormalities. Using an unbiased approach of antibody microarrays it has been found an elevated expression of MMP-1, -2, -3, -8, -10 and -13, in addition to MMP-9, in the tissue from patients with focal cortical dysplasia (Konopka, A. et al., Epilepsy Res., 2013, (1-2):45-58). However, the expression of these proteinases was not as pronounced and/or not as consistent among patients as the expression of MMP-9. Among these other MMPs, especially striking was the upregulation of MMP-2 in adult patients.
Autism spectrum disorders (ASDs) are identified by a cluster of symptoms in three core domains: social interaction, language, and range of interests, but in most cases their etiology is unknown. Fragile X syndrome (FXS) is the leading genetic cause of autism since a large percentage of individuals with FXS (46%) are co-diagnosed with ASD. High plasma activity of MMP-9 has been reported in individuals with FXS (Leigh, M. J. et al., J Dev Behav Pediatr, 2013, (34):1849-1857), whereas elevated protein amounts of MMP-9 were detected in amniotic fluid from ASD mother (Abdallah, M. W. et al., Autism Res, 2012, (5):428-433). Therefore, a clear connection exists between high levels of MMP-9 and ADS.
MMP-9 has been implicated also in human drug addiction, bipolar disorder and depression. One link is provided due to analyses of MMP-9 gene polymorphism at C(-1562)T that is functional, as it results in higher or lower MMP-9 expression. It has been reported that frequently of this polymorphism differentiates between healthy subjects and patients suffering from either bipolar disorders or schizophrenia (Han, H. et al., Psychiatry Res, 2011, (190):163-164). This polymorphism has been linked as well to alcohol addiction (Samochowiec A et al, Brain Res, 2010, 1327:103-6). In addition to this, it has been found that MMP-9 gene polymorphism modulates prefrontal cognition in bipolar men. Increased MMP-9 levels in young patients during bipolar depression have been reported (Rybakowski, J. K., et al., J Affect Disord, 2013, (146):286-289). High levels of MMP-9 in plasma have been detected in depression, this serum levels correlate with the severity of the depression (Yoshida, T. et al., PLoS One, 2012, (7):e42676).
MMP-9 is also claimed to be linked to degenerative diseases such Alzheimer's disease (AD). It has been shown that MMP participates in the formation and clearance of the Aβ peptides in AD. In fact, increased levels of MMP-9 have been observed in the brain tissue and blood of patients with AD, in particular in reactive astrocytes surrounding amyloid plaques, suggesting a local tissue response to plaque accumulation. Several studies have documented that this metalloproteinase participates in Aβ catabolism in vitro and in vivo and it is the only enzyme capable of degrading Aβ fibrils in vitro and Aβ plaques in situ. In addition, it has been reported that MMP-9 is involved in receptor-mediated sAPP-α release and exhibits a α-secretase-like activity in the brain in vivo (Fragkouli, A., et al., J Neurochem, 2012, (121):239-251). Synthetic inhibitors of MMP-2/MMP-9 reduce Aβ-mediated neuronal death in primary cultures (Mizoguchi, H., et al., J Pharmacol Exp Ther, 2009, (331):14-22). In the same study, GM60001 treatment was neuroprotective upon intracerebroventricular administration of Aβ and improved cognition in mice. Moreover, MMP-9 KO mice did not undergo the memory deficits induced by Aβ injections in wild-type mice.
MMP-9 has also been identified to be involved in process and disease conditions other than those related with synaptic plasticity. These conditions include vascular, lung and inflammatory diseases, and cancer. In this regard, MMP-9, and in a lesser extend MMP-2, have been linked to vascular diseases such as ischemic stroke and atherosclerosis, neuropathic pain, inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease, multiple sclerosis, sepsis, cancer, lung diseases such as asthma and chronic obstructive pulmonary disease.
Ischemic stroke is a consequence of a deficit of a blood supply (resulting from a local thrombosis or arterial embolism), producing a decreased tissue oxygenation and in consequence bioenergetics disturbances that may lead to cell death of both necrotic and apoptotic character. Restoration of blood circulation after temporary hypoperfusion results in a robust inflammatory response that may exacerbate the tissue damage. The accumulation of inflammatory cells is then responsible for the high levels of reactive oxygen and nitrogen species as well as proinflammatory cytokines in the ischemic tissue. A dramatic increase of MMP-9 at all levels of its expression and activity is a hallmark of stroke consequences. MMP-9 is involved in such post-stroke events as long-term plasticity, vascular reorganization and angiogenesis, immune response and inflammatory. Blocking the MMPs, MMP-9 in particular, including KO mice, has been demonstrated to be protective against ischemic stroke and its consequences (Chaturvedi, M., et al., Mol. Neurobiol., 2014, (49):563-573)
Treatment of neuropathic pain, triggered by multiple insults to the nervous system, is a clinical challenge because the underlying mechanism of neuropathic pain development remains poorly understood. However, recent studies report that early- and late-phase neuropathic pain development in rats and mice after nerve injury require different MMP. After spinal nerve injury, MMP-9 shows a rapid and transient upregulation in injured dorsal root ganglion (DRG) primary sensory neurons consistent with an early phase of neuropathic pain, whereas MMP-2 shows delayed response in DRG satellite cells and spinal astrocytes consistent with a late phase of neuropathic pain. Intrathecal administration of MMP-9 inhibitors or TIMP-1, an endogenous tissue inhibitor of MMP-9, delay the development of mechanical allodynia (central pain sensitization following painful stimulation) the first days (<10) after the injury. However, the inhibition of MMP-9 has no effect on allodynia when given 10 days after brain injury, showing the critical role of MMP-9 in the early development of neuropathic pain. Compared to MMP-9, MMP-2 upregulation after spinal nerve injury shows a delayed pattern. Intrathecal administration of TIMP-2, an endogenous inhibitor of MMP-2, or small synthetic inhibitors of MMP-2 partly attenuates allodynia on day 1 after injury but almost completely block allodynia in the following ten days. This shows the involvement of MMP-2 in the late phase of neuropathic pain. (Kawasaki, Y., et al., Nature Medicine, 2008, (3):331-336).
During an inflammatory response, leukocyte traffic through tissue barriers, including basement barriers membranes, is only possible if these cells are equipped with enzymes that can remodel ECM. MMP are therefore crucial effector molecules of inflammatory cells. MMPs can act as switches or as delicate turners in acute and chronic inflammation, during autoimmune diseases, when triggered in vascular diseases and in the regenerative phase after inflammation. Thus, MMP biology is important in the definition, execution and resolution phases of acute and chronic inflammatory and ischemic processes and consequently, MMP inhibitors might interfere with these. MMP inhibitors have been tested in many animal models of acute and chronic inflammation, such endotoxin shock, multiple sclerosis and rheumatoid arthritis. Bacteraemia, septic and endotoxin shock are the most frequent causes of mortality in modern hospitals. Bacterial cell-wall constituents induce a systematic response by the activation of the toll-like receptors, leading to an excessive production of inflammatory cytokines and enzymes. Mice deficient in MMP-9 have an altered resistance bacterial induced toxicity, whereas mice deficient in protease inhibitors are more susceptible to endotoxin shock.
Multiple sclerosis is a multifactorial disease that is influenced by genetic predisposition, environmental factors and immunological effector mechanisms that damage the central nervous system. MMP-9 is an immune effector molecule in multiple sclerosis (Opdenakker, G., et al., Lancet Neurol., 2003, (2):747-756). It functions in cell migration through connective tissues and vessel walls and damages the blood-brain barrier. It also lyses protein substrates, such as myelin proteins, cell-adhesion molecules, cytokines and chemokines that are relevant in multiple sclerosis and other neurological diseases. Evidences which supports a detrimental role of MMP-9 in inflammatory CNS damage has been obtained in animal models. Murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, both gelatinases MMP-2 and MMP-9, become upregulated during the development of the disease syndrome (Gijbels, k., et al., J. Neurosci. Res., 1993, (36):432-440). Young MMP-9 deficient mice have resistance to the development of EAE. Double Mmp2/Mmp9-knockout mice are completely resistant against the development of EAE. Pharmacological inhibition of MMP activity improves the course of EAE in several studies that used MMP inhibitors (Hewson, A., et al., Inflamm. Res., 1995, (44):345-349). The use of MMP inhibitors might be also useful in the therapy of rheumatoid arthritis. MPP-9 is involved in the degradation of collagen II during rheumatoid arthritis, leading to the exposure and release of immunodominant epitopes. In addition, MMPs are important for the migration of inflammatory leukocytes. This suggests that MMP inhibitors might be useful in the therapy of rheumatoid arthritis, a notion that has been confirmed in different animal models (Agrawal, S., et al., J. Exp. Med., 2006, (203):1007-1019).
Atherosclerosis and related diseases, including myocardial infarction and stroke, have often been compared with chronic inflammatory diseases. This is based on histopathological findings such as the activation of foamy macrophages, the local production of cytokines and chemokines, and the involvement of MMPs. The use of animal models with genetically altered mice (both transgenic and knockout) has strengthened the view that MMPs are key players in vascular pathologies (Janssens, S., et al., Cardiovsac. Res., 2006, (69):585-594 and Tayebjee, M. H., et al., Curr. Med. Chem., 2005, (12):917-925). It has been reported that MMP-9 levels increase with the progression of idiopathic atrial fibrillation (Li, M. et al., J. Int. Med. Res., 2014, (1):224-230) and is associated with the development of aortic aneurysms (Newman, K. M., et al., Arteriosclerosis and thrombosis: a journal of vascular biology, 1994, (8):1315-1320). Inhibition of MMP-9 suppresses the growth of aortic aneurysms (Lindeman, J. H., et al., Circulation, 2009, (119):2209-2216). Sudden death after myocardial infarction can occur by cardiac rupture, a process in which MMPs are involved. In mouse studies, the critical role of gelatinases in balance with endogenous tissue inhibitors of metalloproteases (TIMPs) is demonstrated. Myocardial infarction could be reversed by treating mice with an oral inhibitor of MMP-2 (Matsumura, S., et al., J. Clin. Invest., 2005, (115):559-609).
MMP-9 has a key role in the pathogenesis of chronic inflammatory disease, including ulcerative colitis and Crohn's disease (Abraham, C., et al., N Eng J Med., 2009, (361):2066-2078), and its upregulation in colonic tissues has been shown to coincide with active flares of inflammatory bowel disease in humans (Gao, Q., et al., Dig Liver Dis., 2005, (37):584-592). In agreement with data from human specimens, it has been observed elevated MMP-2 and MMP-9 protein expression and activation in inflamed colonic tissue in mouse models of inflammatory bowel disease. Moreover, it has been shown that colitis is attenuated in MMP-9 knockout mice, as well as MMP-2 and MMP-9 double-knockout mice. Thus, concomitant inhibition of MMP-2 and MMP-9 is therapeutically effective in inflammatory bowel disease (Grag, P., et al., Am J Physiol Gastrointest Liver Physiol., 2009, (284): 15353-15357).
Mortality in cancer is primarily because of failure to prevent metastasis. Emerging evidences has emphasized the role of MMPs in early aspects of cancer dissemination (Kessenbrock, K., et al., Cell, 2010, (141):52-67). Enzymes that degrade the ECM have long been viewed as essential for tumor progression. Tumor cells are envisioned to produce enzymes that destroy the matrix barriers surrounding the tumor, permitting invasion into surrounding connective tissues, entry and exit from blood vessels, and metastasis to distant organs. MMPs have the capacity to degrade all structural components of ECM. Moreover, MMPs are up-regulated in virtually all human and animal tumors as well as in most tumor cell lines (Coussens, L. M., et al., Science, 2002, (295):2387-2392). MMP-9 is linked to cancer invasion. Elevated levels of MMP-9 in tissue and blood are observed in cancer patients, thus making MMP-9 attractive targets for cancer therapy, since the ability of MMP-9 to degrade collagen and laminin correlates with its ability to regulate cell migration, increase angiogenesis and tumor growth (Bjorklund, M., et al., Biochim Biophys Acta, 2005, (1755):37-69).
In pathological lung conditions, MMPs and their physiological inhibitors (TIMPs) are abnormally over expressed and produced in the respiratory tract by a panel of different structural cells. Alternations in these biological activities have several dramatic effects in wound healing and cell trafficking. Deregulation of various MMPs by stimulated structural or inflammatory cells is thought to take part to pathophysiology of numerous lung diseases including asthma, chronic obstructive pulmonary disease (COPD), lung fibrosis and lung cancer (Demedts, I. K., et al., Curr Opin Pharmacol, 2005, (5):257-63). The inflammation process is characterized by the extracellular matrix remodeling and collagen deposition that in turns request increased levels of MMP-9 (Kelly, E. A., et al., Am J Respir Crit Care Med, 2000, (162):1157-1161). Selective inhibition of MMP-9 is thought to promote a therapeutic benefit in these associated chronic inflammatory lung diseases as it has been proven recently in the treatment of COPD (Xie, S. S., et al., J Int Med Res, 2014, (42):1272-1284).
According to these experimental evidences, there is an existing need of finding new compounds which would be potent inhibitors of MMP-2 and MMP-9 and would be selective with respect to other MMPs such as collagenases (MMP-1), stromlysins (MMP-3) and matrylisins (MMP-7).